Gene therapy offers the prospect of selectively introducing genes into cancer cells, leaving them susceptible to specific antitumor drugs. Current protocols to elicit tumor reduction utilize Herpes Simplex Virus type 1(HSV) thymidine kinase (TK) with the prodrug ganciclovir (GCV) or the E. coli cytosine deaminase with the prodrug 5-fluorocytosine (5FC). While the wild-type TK is functional as a suicide gene, a number of caveats restrict its full effectiveness. These include a poor Km or binding affinity for GCV (approximately 47muM) and the toxicity associated with high doses of GCV. Another prodrug, acyclovir (ACV), predominantly used as an anti-herpetic drug, does not demonstrate the immunosuppressive attributes of GCV, even at very high doses. However, the very high Km that HSV TK displays towards ACV (approximately 320 muM), precludes its use as a prodrug in ablative gene therapy. Similarly, there are caveats with cytosine deaminase and 5FC that preclude its potential as a safe and effective suicide gene. We seek to identify the optimal suicide gene and prodrug combination for the safest and most effective cancer gene therapy. Towards this end we seek 1) to understand the structure-function relationship of nucleoside metabolizing enzymes important to suicide gene therapy and 2) to manipulate HSV-1 thymidine kinase and cytosine deaminase genes for superior performance in ablative gene therapy settings. The goal of this work is to 1) create novel genes by mutagenesis, 2) evaluate mutant genes for improved tumor sensitivity to various prodrugs, 3) construct mini-pathways and 4) create fusion proteins with other genes to enhance prodrug activation and tumor ablation. Not only will the results from the project impact the choice of gene(s) used for cancer treatment, but it also has wide-reaching applications including graft versus host disease, restenosis, AIDS, tumor imaging, cell lineage ablation studies, in negative selection systems and selection against non-homologous recombination for the generation of transgenic mice. Furthermore, understanding the molecular basis of nucleoside metabolizing enzyme function and interaction with current drugs will have far-reaching ramifications in the design, development and use of novel antiviral, antifungal and antibacterial drugs.